Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons in the central nervous system. Degeneration of the motor neurons results in paralysis and eventual death, usually due to respiratory failure. In a subset of cases, ALS is caused by dominantly transmitted mutations in the gene encoding cytosolic superoxide dismutase (SOD1). Transgenic expression of mutant SOD1 causes ALS in mice. In these mice, suppression of expression of the transgene using either antisense oligonucleotides or siRNA slows progression and improves survival. While siRNA-based drugs represent a potential therapeutic paradigm for the treatment of CNS and other disorders, the ability to apply this technology to human disease has been impeded by the absence of efficient and non-toxic in vivo delivery systems. We have recently developed a novel class of covalently modified RNAi compounds that do not require a delivery vehicle to enter cells and have improved pharmacology compared to traditional siRNAs. We term these compounds "self-delivering rxRNA" or sd-rxRNATM. sd-rxRNA is a hydrophobically modified RNAi-antisense hybrid, which has been demonstrated to be highly efficacious in vivo upon local administration. Robust silencing without toxicity has been demonstrated after intradermal and intravitreal injection, where ug doses induce potent and long lasting silencing. We have already identified an sd-rxRNA compound targeting the SOD1 gene and have demonstrated delivery to spinal cord using implanted intrathecal pumps. Intrathecal administration is a clinically relevant delivery mode that has been used previously for ALS. The objective of this proposal is to optimize and characterize sd-rxRNA technology for gene silencing in spinal cord and development of SOD1 targeting therapeutics for treatment of ALS. PUBLIC HEALTH RELEVANCE: Narrative RNAi (RNA interference) has the potential to treat human disease by down-regulation of disease causing genes. However, efficient delivery is a major road block for therapeutic development of RNAi. We have recently developed chemically modified RNAi compounds that enter cells in vivo ("self-delivering" RNAi compounds). This project will explore the use of these compounds in spinal cord delivery, in particular for silencing of SOD1 in familial cases of ALS.